EP2183384B1 - Procédé et dispositif destinés à la détermination de la concentration d'un analyte par mesure de la fluorescence - Google Patents

Procédé et dispositif destinés à la détermination de la concentration d'un analyte par mesure de la fluorescence Download PDF

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Publication number
EP2183384B1
EP2183384B1 EP08785201.8A EP08785201A EP2183384B1 EP 2183384 B1 EP2183384 B1 EP 2183384B1 EP 08785201 A EP08785201 A EP 08785201A EP 2183384 B1 EP2183384 B1 EP 2183384B1
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EP
European Patent Office
Prior art keywords
fluorophore
fluorescence
concentration
glucose
lifetime
Prior art date
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Not-in-force
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EP08785201.8A
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German (de)
English (en)
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EP2183384A1 (fr
Inventor
Wolfgang Petrich
Hans-Peter Haar
Joachim Hoenes
Carina Horn
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose

Definitions

  • the invention relates to a method for determining the concentration of glucose in a liquid sample.
  • it is about the examination of medical samples, especially of blood and / or interstitial fluid on analytes contained therein.
  • the invention is directed to methods in which the determination of the concentration of the analyte is based on a fluorescence measurement.
  • a reagent system is used whose reaction with the sample results in a change in the amount of a fluorophore, which change in amount may in principle be both an increase by formation of a fluorophore and a decrease by consumption of a fluorophore.
  • the reaction system is chosen so that the change in the amount of fluorophore for the desired analytical concentration is characteristic.
  • the analysis method includes measuring a quantity correlated with the amount of the fluorophore. On the basis of the resulting measured values, the concentration is determined by means of an evaluation algorithm.
  • NADH nicotinamide adenine dinucleotide
  • the invention is based on the technical problem of proposing a method which enables improved measurement accuracy, in particular with regard to the mentioned measurement errors and disturbances with reduced effort. It should be particularly suitable for small appliances that work as easily and inexpensively.
  • the subject of the invention is also an analysis system for carrying out the method.
  • the fluorescence lifetime surprisingly depends on the analyte concentration to such an extent that measured values of this measurand are advantageously used in the algorithm for determining the analytical result (desired concentration) can be used.
  • this can be explained as follows for the case of a glucose test with the reagent system GlucDH / NAD.
  • NADH formed as a result of the reaction of glucose with the enzyme-coenzyme pair GlucDH / NAD forms a complex with GlucDH.
  • This complex formation affects the fluorescence lifetime of NADH: The fluorescence of the free NADH is short-lived because molecules in the surrounding fluid act as quenchers. In the complex, the NADH molecule is largely protected against the action of the quenchers and the fluorescence is much more durable.
  • the fluorescence lifetime provides second, independent information about the fluorescence of the reaction products in addition to the fluorescence intensity. It can be used independently (ie without other information) to determine the analyte concentration sought. Preferably, however, it is used in combination with the measurement of fluorescence intensity. This requires no additional metrological effort, because conventional methods for measuring the fluorescence lifetime at the same time provide measurement results on the fluorescence intensity.
  • the fluorescence lifetime may be expedient to measure it and to take the measured values resulting from the measurement into consideration in the evaluation algorithm in a suitable manner.
  • a measurement in the strict sense, ie measurement of previously unknown measured values
  • the fluorescence lifetime may also be used in a manner that does not require measurement in the sense that fluorescence lifetime (previously unknown) measurements are determined.
  • One possibility is to measure the fluorescence intensity within a defined time window, the time window corresponding to the (known) fluorescence lifetime adapted from the reaction of the sample with the reagent system resulting fluorophore.
  • the measurement value resulting from such a measurement is related to the fluorescence lifetime and is hereinafter also referred to as lifetime related fluorescence intensity. Since it is used in the evaluation algorithm, it can also be said in this case that the fluorescence lifetime is used in the evaluation algorithm.
  • the evaluation device of a corresponding analysis system since measurement of the fluorescence lifetime (in the strict sense explained) is not absolutely necessary, the evaluation device of a corresponding analysis system also does not necessarily have to have a measuring unit which is designed to measure the fluorescence lifetime of the fluorophore. Rather, alternatively or additionally, there is the possibility that the measuring unit of the evaluation device is designed such that it takes into account the fluorescence lifetime in a different manner, for example by means of the time window mentioned in the intensity measurement.
  • the analysis system shown in highly schematic form consists of two matched components, namely an analysis element 2 and an evaluation device 3.
  • the analysis element 2 comprises a reagent layer 4 which contains a reagent system in a suitable matrix, for example a gel matrix.
  • the reagent layer 4 is located on an optically transparent support 5.
  • the reagents of the reagent system are embedded in the matrix of the reagent layer 4 in dry form.
  • a sample liquid 6 is applied to and enters the free surface of the reagent layer 4
  • the reagents of the reagent system are dissolved and a reaction of the sample liquid occurs which results in a change in the amount of a fluorophore which is part of the reagent system.
  • the structure of such analysis elements for example, in the documents US 3,802,842 ; U.S. 4,061,468 ; US 2006/0003397 described.
  • the housing 7 of the evaluation device 3 contains a total of 8 designated measuring unit, which is suitable to determine a characteristic of the amount of the fluorophore measurement.
  • the measuring unit 8 includes an excitation light source 10, for example a laser or an LED whose light is directed from behind (through the optically transparent support 5) to the surface of the reagent layer 4 facing away from the sample application side and facing the optically transparent support 5.
  • an excitation light source 10 for example a laser or an LED whose light is directed from behind (through the optically transparent support 5) to the surface of the reagent layer 4 facing away from the sample application side and facing the optically transparent support 5.
  • secondary light is radiated from the evaluation zone 9 in the direction of a detector 11 and detected by this.
  • the resulting measurement signal is fed to a signal processing unit and amplified, processed and digitized in the usual way.
  • the resulting digitized measured values are fed to an evaluation unit 13 which contains the required software and hardware in order to determine the sought-after concentration of the analyte from
  • the measuring unit 8 is suitable for detecting fluorescent light emitted by the evaluation zone 9.
  • the access of the emanating from the excitation light source 10 primary light blocked such that the radiated with a higher wavelength fluorescent light can be selectively detected by the detector 11.
  • the analysis system of the invention is conventional and therefore need not be explained in detail. Further information can be found, for example, in the cited documents of the prior art.
  • a peculiarity of in FIG. 1 illustrated analysis system according to the invention is that the measuring unit 8 is adapted to measure the mean fluorescence lifetime and / or to take into account (in the context of the measurement of another measure).
  • the fluorescence lifetime measurement is preferably carried out using a phase modulation method.
  • the light emitted by the excitation light source 10 is high-frequency modulated, and the sought information about the fluorescence lifetime is determined from the phase shift measured by the detector 11 and the signal processing unit 12.
  • a more detailed explanation is not required because additional information in the literature, in particular the documents mentioned above, can be found.
  • the measurement results shown are derived from the fact that the change in the mean lifetime can be used as information for the quantification of an analyte in fluorescence tests. This insight can be used analytically in different ways.
  • FIG. 3 illustrates a preferred approach in which the fluorescence lifetime measurement is used to determine a lifetime-related fluorescence intensity (LRFI).
  • LRFI lifetime-related fluorescence intensity
  • Such an LRFI value describes the intensity of the fluorescence as a function of the fluorescence lifetime and thus forms a measure of the quantitative ratio of fluorescent species which are distinguished by different average lifetimes, such as, for example, complexed NADH in relation to free NADH.
  • an LRFI value for example, by the decay of the fluorescence signal being a logarithmic function, which depends on the mean fluorescence lifetimes of the fluorescent species and their concentration.
  • the separated intensities of the two signal components can be determined by mathematically fitting such traces.
  • FIG. 3 shows the dependence of such LRFI values on the glucose concentration for a glucose fluorescence assay with analysis elements as described in the above-mentioned documents (2) and (3). Shown is a ratio R s / l between the LRFI value for short-lived free NADH and the LRFI value for long-lived complexed NADH.
  • the measurements were carried out with blood samples of different hematocrit values and with an aqueous glucose solution, the resulting measured values in FIG. 3 are denoted by different symbols.
  • # 1 to # 3 denote the numbers of samples having different hematocrit concentrations (very high - normal range - very low); "ags" means "aqueous glucose solution”. you recognizes an astonishingly good correlation between the glucose concentration and the R sl value, the measured values being largely independent of the hematocrit value, that is to say the concentration of the blood corpuscles in the sample.
  • FIG. 3 shows a first example of a preferred embodiment of the invention in which two LRFI values are related.
  • the value R s / l is calibrated against the analyte concentration and then used in the evaluation algorithm as a measure of the analyte concentration.
  • fluorescence lifetimes and the fluorescence intensities of two fluorescent species are used as characteristic parameters for the analysis. They are each set in relation to one another according to a predefined mathematical equation in order to determine an arithmetic variable which can be calibrated against the analyte concentration sought.
  • the use of two simultaneously measured values and the formation of a quotient results in measurement errors which affect both fluorescence measured values in the same way, for example due to contamination or small damages in the optical measuring channel, not or only to a much lesser extent negatively influencing the measurement result than in prior art measurements of fluorescence intensity.
  • LRFI values are an example of the fact that measurement of a fluorescence lifetime (in the strict sense) is not necessarily required in the context of the invention.
  • the measuring device of the evaluation device can also be designed such that it detects a fluorescence intensity of a defined time window and thus an LRFI value is measured, preferably two or more such LRFI values, similar to those explained in the preceding examples, be set in relation to each other to determine a computational variable that can be calibrated against the analyte concentration.
  • FIG. 4 shows the correlation of glucose concentration values C m determined according to the invention with glucose values C ref , which were determined using a reference method.
  • the fluorophore is a coenzyme selected from the group consisting of NADH / H + and NADPH / H + , including their derivatives.
  • Particularly suitable are derivatives in which the part of the molecule which is essentially responsible for the fluorescence remains unchanged. In such derivatives, basically analogous fluorescence properties are to be expected, whereby, of course, the parameters of the fluorescence, such as the wavelength of the absorption or emission, may change. Since in NAD / NADH essentially the pyridine ring is responsible for the fluorescence, in particular derivatives are suitable in which the pyridine ring is not modified.
  • suitable derivatives are, for example, in WO2007 / 012494 and the US2007 / 26476 described.
  • carbaNAD a derivative without glycosyl bond, which has already been described in 1988, is suitable for use in the process according to the invention ( JT Slama, Biochemistry 1989, 27, 183 and Biochemistry 1989, 28, 7688 ).
  • the ribose is substituted by a carbacyclic sugar unit.
  • not all derivatives of NADH / H + and NADPH / H + are equally suitable for the invention.
  • the suitability for the use according to the invention can easily be tested experimentally.
  • the enzyme of an enzyme / coenzyme pair used in the invention is glucose dehydrogenase (EC1.1.1.47). eg L-amino acid dehydrogenase.
  • the average fluorescence lifetime correlates so well with the concentration of the analyte that it can be used independently as the characteristic quantity for the concentration in the analyte concentration analysis algorithm.
  • it is preferably used in combination with another measurement variable characterizing the concentration, in particular the fluorescence intensity, but also the optical absorption. It can also be used to compensate for measurement errors caused by disturbances, in particular the temperature of the sample or its hematocrit.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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Claims (9)

  1. Procédé de détermination de la concentration de glucose dans un échantillon liquide au moyen d'un système de réactifs qui contient une paire enzyme/coenzyme avec une glucose déshydrogénase (GlucDH) en tant qu'enzyme et de la NAD et/ou du NADP, y compris leurs dérivés appropriés pour lesquels la part moléculaire essentiellement responsable de la fluorescence n'est pas modifiée, en tant que coenzyme,
    dans lequel,
    la réaction de l'échantillon avec le système de réactifs conduit à une modification de la quantité d'un fluorophore qui est sélectionné parmi le groupe constitué de NADH/H+ et NADPH/H+, y compris leurs dérivés appropriés pour lesquels la part moléculaire essentiellement responsable de la fluorescence n'est pas modifiée,
    la quantité du fluorophore est corrélée à la concentration du glucose,
    une valeur caractéristique de la quantité du fluorophore est mesurée et
    la concentration du glucose est déterminée au moyen d'un algorithme d'évaluation sur la base de ladite valeur,
    caractérisé en ce que
    le système de réactifs est intégré dans un élément d'analyse sous une forme sèche,
    la réaction de l'échantillon avec le système de réactifs inclut une formation de complexes avec participation d'une paire enzyme/coenzyme, dans lequel l'enzyme est la GlucDH et le coenzyme est le fluorophore, et
    la modification, liée à la formation de complexes, de la durée de vie moyenne de fluorescence du fluorophore est utilisée lors de la détermination de la concentration du glucose.
  2. Procédé selon la revendication 1, caractérisé en ce que le fluorophore est un dérivé du NADH/H+ ou du NADPH/H+, dont le cycle pyridine n'est pas modifié, ou est une carbaNAD.
  3. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que la durée de vie moyenne de fluorescence mesurée du fluorophore est utilisée dans l'algorithme d'évaluation en tant que valeur caractéristique de la concentration.
  4. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que la durée de vie moyenne de fluorescence mesurée est utilisée dans l'algorithme d'évaluation en combinaison avec une autre valeur caractéristique de la concentration, en particulier l'intensité de fluorescence.
  5. Procédé selon la revendication 4, caractérisé en ce que la durée de vie moyenne de fluorescence mesurée est utilisée dans l'algorithme d'évaluation pour compenser des erreurs de mesure provoquées par des perturbations.
  6. Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que la mesure de la durée de vie moyenne de fluorescence est utilisée pour déterminer une valeur d'intensité de fluorescence en lien avec la durée de vie.
  7. Procédé selon la revendication 6, caractérisé en ce que l'intensité de fluorescence est mesurée pour au moins deux durées de vie moyennes de fluorescence différentes, afin de déterminer respectivement des valeurs d'intensité de fluorescence en lien avec la durée de vie.
  8. Procédé selon la revendication 7, caractérisé en ce que les deux valeurs d'intensité de fluorescence en lien avec la durée de vie sont mises en relation l'une par rapport à l'autre.
  9. Système d'analyse destiné à déterminer la concentration de glucose dans un échantillon liquide au moyen d'un procédé selon l'une quelconque des revendications précédentes,
    avec
    un système de réactifs qui contient une glucose déshydrogénase (GlucDH) en tant qu'enzyme et de la NAD et/ou du NADP, y compris leurs dérivés appropriés pour lesquels la part moléculaire essentiellement responsable de la fluorescence n'est pas modifiée, en tant que coenzyme,
    dont la réaction avec l'échantillon conduit à une modification de la quantité d'un fluorophore,
    - qui est sélectionné parmi le groupe constitué de NADH/H+ et NADPH/H+, y compris leurs dérivés appropriés pour lesquels la part moléculaire essentiellement responsable de la fluorescence n'est pas modifiée,
    - dans lequel la quantité du fluorophore est corrélée à la concentration du glucose,
    - dans lequel la réaction de l'échantillon avec le système de réactifs inclut une formation de complexes avec participation d'une paire enzyme/coenzyme, dans lequel l'enzyme est la GlucDH et le coenzyme est le fluorophore, et
    un appareil d'évaluation, qui inclut une unité de mesure destinée à mesurer une valeur caractéristique de la quantité du fluorophore et qui inclut une unité d'évaluation pour déterminer la concentration du glucose au moyen d'un algorithme d'évaluation sur la base de ladite valeur,
    caractérisé en ce que
    les réactifs du système de réactifs sont contenus sous une forme sèche dans un élément d'analyse, et
    l'unité de mesure de l'appareil d'évaluation est conçue pour mesurer et/ou considérer une modification, liée à la formation de complexes, de la durée de vie moyenne de fluorescence du fluorophore.
EP08785201.8A 2007-08-01 2008-07-30 Procédé et dispositif destinés à la détermination de la concentration d'un analyte par mesure de la fluorescence Not-in-force EP2183384B1 (fr)

Priority Applications (1)

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EP08785201.8A EP2183384B1 (fr) 2007-08-01 2008-07-30 Procédé et dispositif destinés à la détermination de la concentration d'un analyte par mesure de la fluorescence

Applications Claiming Priority (3)

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EP07015084A EP2022859A1 (fr) 2007-08-01 2007-08-01 Procédé et dispositif destinés à la détermination de la concentration d'un analyte par mesure de la fluorescence
EP08785201.8A EP2183384B1 (fr) 2007-08-01 2008-07-30 Procédé et dispositif destinés à la détermination de la concentration d'un analyte par mesure de la fluorescence
PCT/EP2008/006255 WO2009015870A1 (fr) 2007-08-01 2008-07-30 Procédé et dispositif pour déterminer la concentration d'un analyte par mesure de la fluorescence

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EP2183384A1 EP2183384A1 (fr) 2010-05-12
EP2183384B1 true EP2183384B1 (fr) 2016-03-16

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EP08785201.8A Not-in-force EP2183384B1 (fr) 2007-08-01 2008-07-30 Procédé et dispositif destinés à la détermination de la concentration d'un analyte par mesure de la fluorescence

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EP1504116A1 (fr) * 2002-05-16 2005-02-09 F. Hoffmann-La Roche AG Procede et systeme de reactifs contenant une enzyme inactivee
MX2011009387A (es) * 2009-03-20 2011-09-28 Hoffmann La Roche Elemento de prueba para analizar un fluido corporal y metodo para su medicion.
JP6059244B2 (ja) * 2011-11-14 2017-01-11 エフ ホフマン−ラ ロッシュ アクチェン ゲゼルシャフト 試料中の少なくとも１つの分析物を検出するための分析装置
EP2620507A1 (fr) 2012-01-25 2013-07-31 Roche Diagnostics GmbH Procédé pour l'évaluation de la qualité d'un élément de test
CN105636634B (zh) 2013-09-30 2019-02-01 圣犹达医疗用品心脏病学部门有限公司 具有主动回直机构的导管
CA3035874A1 (fr) 2016-10-05 2018-04-12 F. Hoffmann-La Roche Ag Reactifs de detection et agencements d'electrodes pour elements de test de diagnostic multi-analytes, ainsi que leurs procedes d'utilisation
CN113176239B (zh) * 2021-04-27 2023-04-14 国家纳米科学中心 一种确定荧光多肽自组装临界组装浓度的方法及其应用

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EP1504116A1 (fr) * 2002-05-16 2005-02-09 F. Hoffmann-La Roche AG Procede et systeme de reactifs contenant une enzyme inactivee
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US20100227348A1 (en) 2010-09-09
EP2022859A1 (fr) 2009-02-11
WO2009015870A1 (fr) 2009-02-05
EP2183384A1 (fr) 2010-05-12

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